Synergistic effect of isoxaben and cellulosin as an herbicide

ABSTRACT

The present application relates to a method of weed control for a crop field using a synergistic effect of two herbicides cellulosin and isoxaben. A composition matter together with said method comprising cellulosin and isoxaben is within the scope of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application is a divisional of U.S. application Ser.No. 16/559,799, filed Sep. 4, 2019, and relates to and claims thepriority benefit of U.S. Provisional Application Ser. No. 62/726,443,filed Sep. 4, 2018, the contents each of which is hereby incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present application relates to a method for weed control of a fieldusing the synergistic effect of two or more herbicides, includingcellulosin and isoxaben. A composition matter comprising cellulosin andisoxaben is also in the scope of the present invention.

BACKGROUNDS AND SUMMARY OF THE INVENTION

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

Weed plants compete with desired crops in the field for resources andoften negatively affect crop yield and quality significantly (Soltani,N, et al., Weed Technol. 2016, 30, 979-984; Swanton, C. J., et al., WeedTechnol., 1993, 7, 537-542; Soltani, N, et al., Weed Technol. 2018, 32,342-346). Herbicides play essential roles in agricultural productions inboth developed and developing countries by allowing farmers to controlweed plants without using a lot of human labor (Gianessi, L. P. PestManag Sci. 2013, 69, 1099-1105). By growing herbicide-resistant cropsthat are created through genetic modification and spraying correspondingherbicide in the field, the farmers can control weed plants withoutkilling the crops (Funke, T, et al., Proc Natl Acad Sci USA 2006, 103,13010-13015). However, herbicide-resistant weeds have become asignificant problem in agricultural production because of repetitiveapplication of the same type of herbicide (Edwards, C. B., et al., PestManag. Sci. 2014, 70, 1924-1929; Beres, Z. T. et al., Sci Rep. 2018, 8,10483; Morran, S. et al., Pest Manag Sci, 2018, 74, 2747-2753).Developing herbicide with a novel mode of action and crops that areresistant to the novel herbicide is essential for efficient weed controlin global agricultural production.

A method is disclosed herein for weed control of a crop field using thesynergistic effect of two different herbicides cellulosin and isoxaben.

A method is disclosed herein for weed control of a crop field using acombination of cellulosin and isoxaben, together with one or more otherherbicides of the same or different mode of action.

A method is disclosed herein for reducing the resistance of a weed to anherbicide, wherein the method comprises the step of treating a weed witha combination of cellulosin and isoxaben, together with one or morediluents, excipients or carriers.

A composition for weed control of a crop field comprises cellulosin andisoxaben.

BRIEF DESCRIPTION OF DRAWINGS

The disclosed embodiments and other features, advantages, anddisclosures contained herein, and the matter of attaining them, willbecome apparent and the present disclosure will be better understood byreference to the following description of various exemplary embodimentsof the present disclosure taken in conjunction with the accompanyingdrawings, wherein:

FIGS. 1A-1F show that known isoxaben resistant mutants ixr1-1, ixr1-2and ixr2-1 are not resistant to Cellulosin. Wildtype (WT) Arabidopsisand its mutants ixr1-1, ixr1-2, and ixr2-1 are grown on mediasupplemented with DMSO (FIGS. 1A-1B), isoxaben (FIGS. 1C-1D) orCellulosin (FIGS. 1E-1F) and their quantifications on their root length.Arabidopsis mutants ixr1-1, ixr1-2 and ixr2-1 have longer roots incompare with wildtype (Col) on media supplemented with isoxaben.However, these mutants do not show difference in growth in compare withwildtype (Col) on media supplemented with Cellulosin. Bars: 1 cm

FIGS. 2A-2D show that, as compared with DMSO control (FIG. 2A), most ofCellulosin resistant mutants are sensitive to compounds isoxaben (FIG.2B), indaziflam (FIG. 2C) and C17 (FIG. 2D).

FIGS. 2E-2G depict quantifications of root length of Cellulosinresistant mutants on media supplemented with isoxaben (FIG. 2E),indaziflam (FIG. 2F), and C17 (FIG. 2G) in comparison with DMSO control,Statistically significant differences in right panels were determined byone-way ANOVA test followed by Tukey's multiple comparisons test.Different letters of root length of seedlings indicate significantdifference between groups (p<0.05). Bars: 1 cm.

FIGS. 3A-3D demonstrate that plants expressing CesA6 carrying mis-sensemutations at the catalytic site of Cellulosin do not show resistance toisoxaben, indaziflam and C17 in growth compared with DMSO control: DMSO(FIG. 3A), isoxaben (FIG. 3B), Indaziflam (FIG. 3C) and C17 (FIG. 3D).Bars: 1 cm.

FIGS. 4A-4B demonstrate the synergistic effect of Cellulosin withisoxaben and indaziflam. FIG. 4A shows Arabidopsis wildtype plants grownon DMSO, Cellulsoin, isoxaben, indaziflam, C17, Cellulosin withisoxaben, Cellulosin with indaziflam, and Cellulosin with C17. Plantgrowth was not inhibited by 0.25 μM Cellulosin, 4 nM isoxaben, 0.25 nMindaziflam or 4 nM C17 significantly. However, combined application of0.25 μM Cellulosin and 4 nM isoxaben, 0.25 μM Cellulosin and 0.25 nMindaziflam, or 0.25 μM Cellulosin and 4 nM C17 significantly inhibitedplant growth. Bar, 1 cm.

FIG. 4B shows quantification on the root length of Arabidopsis plantsgrown on media with control (0.1% DMSO), 0.25 μM Cellulosin, 4 nMisoxaben, 0.25 nM indaziflam, 4 nM C17, 0.25 μM Cellulosin and 4 nMisoxaben, 0.25 μM Cellulosin with 0.25 nM indaziflam, or 0.25 μMCellulosin and 4 nM C17. Statistically significant differences in rightpanels were determined by one-way ANOVA test followed by Tukey'smultiple comparisons test. Different letters of root length of seedlingsindicate significant difference between groups (p<0.05). N=12.

FIGS. 5A-5F demonstrate that Cellulosin inhibits the growth of differentplant species. FIG. 5A shows Cellulosin inhibition of tomato growth.FIG. 5B shows Cellulosin inhibition of tobacco growth. FIG. 5C showsCellulosin inhibition of Kentucky Bluegrass growth. FIG. 5D showsCellulosin inhibition of Perennial Ryegrass growth. FIG. 5E showsCellulosin inhibition of soybean growth. FIG. 5F shows Cellulosininhibition of maize growth. Bars: 1 cm. Statistical analysis wasperformed using student t-test. ** p<0.01. *** p<0.001.

FIGS. 6A-6B demonstrate that Cellulosin inhibits plant growth in soil.FIG. 6A shows Arabidopsis plants sprayed with control solution. FIG. 6Bshows Arabidopsis plants sprayed with Cellulosin (500 μM).

FIG. 7 demonstrates synergistic effect between Cellulosin and isoxaben.Neither 300 nM Cellulosin nor 3.5 nM isoxaben significantly inhibitsplant growth. However, combined application of 300 nM and 3.5 nMisoxaben significantly inhibits plant growth. Nine days old Arabidopsisplants grown on control condition (0.1% DMSO), 300 nM Cellulosin, 3.5 nMisoxaben, or 300 nM Cellulosin and 3.5 nM Isoxaben are shown. Bar, 1 cm.

An overview of the features, functions and/or configurations of thecomponents depicted in the various figures will now be presented. Itshould be appreciated that not all of the features of the components ofthe figures are necessarily described. Some of these non-discussedfeatures, such as various couplers, etc., as well as discussed featuresare inherent from the figures themselves. Other non-discussed featuresmay be inherent in component geometry and/or configuration.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

In the present disclosure, the term “about” can allow for a degree ofvariability in a value or range, for example, within 20%, within 10%,within 5%, or within 1% of a stated value or of a stated limit of arange.

In the present disclosure, the term “substantially” can allow for adegree of variability in a value or range, for example, within 70%,within 80%, within 90%, within 95%, or within 99% of a stated value orof a stated limit of a range.

In this document, the terms “a,” “an,” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.In addition, it is to be understood that the phraseology or terminologyemployed herein, and not otherwise defined, is for the purpose ofdescription only and not of limitation. Any use of section headings isintended to aid reading of the document and is not to be interpreted aslimiting. Further, information that is relevant to a section heading mayoccur within or outside of that particular section. Furthermore, allpublications, patents, and patent documents referred to in this documentare incorporated by reference herein in their entirety, as thoughindividually incorporated by reference. In the event of inconsistentusages between this document and those documents so incorporated byreference, the usage in the incorporated reference should be consideredsupplementary to that of this document; for irreconcilableinconsistencies, the usage in this document controls.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant comprising the step ofapplying isoxaben and cellulosin, together with one or more diluents,excipients or carriers to a field in need of weed control.

In some other illustrative embodiments, the method of weed control for afield of a plant further comprise a step of applying one or more otherherbicides of the same or different mode of action to said field.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant, wherein said isoxaben andcellulosin are applied to a field of a plant together as a mixture ofpreformulated single product.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant, wherein said isoxaben andcellulosin are applied to a field of a plant separately as anindividually pre-formulated product, consequentially or concurrently.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant, wherein said isoxaben andcellulosin are applied to a field of a plant together with one or moreother herbicides of the same or different mode of action.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant, wherein said plant is acrop for food or feed.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant as disclosed herein,wherein said plant is resistant to cellulosin.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant as disclosed herein,wherein said plant is resistant to isoxaben.

In some illustrative embodiments, this present invention relates to amethod of weed control for a field of a plant as disclosed herein,wherein the ratio of said isoxaben and cellulosin ranges from about1:100 to about 100:1 on a molar basis.

In some illustrative embodiments, this present invention relates to acomposition comprising isoxaben and cellulosin, together with one ormore diluents, excipients or carriers.

In some illustrative embodiments, this present invention relates to acomposition comprising isoxaben and cellulosin as disclosed herein,wherein the ratio of isoxaben and cellulosin ranges from about 1:100 toabout 100:1 on a molar basis.

In some illustrative embodiments, this present invention relates to acomposition comprising isoxaben and cellulosin as disclosed herein,wherein amount/concentration of isoxaben and cellulosin ranges fromabout 0.01 mg/mL to about 5000 mg/mL.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance comprising the step of applyingcellulosin and isoxaben to a field of a plant for weed control therein.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said method furthercomprising a step of applying one or more other herbicides of the sameor different mode of action to a field of a plant for weed controltherein.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said isoxaben andcellulosin are applied to a field of a plant together as a mixture ofpre-formulated single product.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said isoxaben andcellulosin are applied to a field of a plant separately as anindividually pre-formulated product, consequentially or concurrently.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said isoxaben andcellulosin are applied to a field of a plant together with one or moreother herbicides of the same or different mode of action.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said plant isresistant to cellulosin and isoxaben.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein said plant is a cropfor food or feed.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein the ratio of isoxabenand cellulosin ranges from about 1:100 to about 100:1 on a molar basis.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance, wherein the amount of saidisoxaben and cellulosin ranges from about 0.01 mg/mL to about 5000mg/mL.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance as disclosed herein, whereinsaid plant is resistant to cellulosin.

In some illustrative embodiments, this present invention relates to amethod for reducing herbicide resistance as disclosed herein, whereinsaid plant is resistant to isoxaben.

In some embodiments, the composition of isoxaben and cellulosin may beformulated in various dosage forms, including, but not limited to, dryformulation, liquid formulation, granular or pellet formulation. Thepractice and information are known in the arts. In some otherembodiments, the final product of the composition disclosed herein maybe formulated as a suspension, a liquid spray, a powder, a nanoparticle,or an aerosol, together with one or more adjuvants, excipients orcarriers.

In preparing a product for an end user, adjuvants, surfactants,anti-drifting agents, colorings, anti-freezing or other stabilizingchemicals may be included. An adjuvant is an additive (usually inrelatively low amounts compared to the carrier) that improves orenhances application, performance, safety, storage, or handling of anactive ingredient. Adjuvants include materials such as: Surfactants(spreaders, stickers, emulsifiers, wetting agents), which increasesurface contact, reduce runoff, and increase penetration through leafcuticle.

It is understood that, the herbicides disclosed herein can be applied toa field of a plant for weed control at the same time as a pre-formulatedmixture, or applied individually as a separately pre-formulated product,consequentially or concurrently.

It is understood that, multiple application of said composition ofherbicides may be needed in some cases in order achieve effective andefficient weed control for a field of a plant. As disclosed herein saidplant is resistant to the herbicides applied.

Isoxaben (N-3[1-ethyl-1-methylpropyl]-5-isoxazolyl-2,6, ordimethoxybenzamide) is a broad-leaf herbicide used primarily on smallgrains, turf, and ornamentals. It has been suggested that Isoxabeninhibits the incorporation of glucose into the cellulose-richacid-insoluble fraction of isolated cell walls and has been proposed tobe a specific inhibitor of cellulose biosynthesis. Treated cells ofsensitive species fail to elongate normally and consequently growisodiametrically. Analysis of the effects of the compound on thepolysaccharide composition of cell walls and other aspects of plantphysiology has led to the proposal that the herbicidal action ofisoxaben can be explained entirely by its effect on cellulosebiosynthesis.

As it is disclosed herein, cellulosin is a small molecule compound thatacts as an inhibitor toward cellulose synthase (CesA), an enzyme thatcatalyzes the synthesis of cellulose. Cellulosins were discovered aspotential herbicides, which is described in Zhang, U.S. PatentApplication Publication US 2020/0290959 A1, published Sep. 17, 2020,wherein the structures of cellulosins are defined in claim 4 and furtherexemplified by FIG. 1A.

Cellulosin is a small molecule that inhibits plant growth by targetingthe conserved catalytic domain of cellulose synthase (CesA), the enzymethat catalyzes the synthesis of cellulose. The discovery of Cellulosinis being documented in Zhang, U.S. Patent Application Publication, US2020/0290959 A1, published Sep. 17, 2020, and the content of which isincorporated herein in its entirety. Further characterization of theherbicide effect of Cellulosin by spraying the Arabidopsis plants withCellulosin and testing the inhibitory effects of Cellulosin on differentcrops led to our discovery that Cellulosin has different mode of actionthan other cellulose synthesis inhibitors isoxaben, indaziflam and C17.The known mutants that are resistant to isoxaben are sensitive toCellulosin. Most of our mutants that are resistant to Cellulosin aresensitive to isoxaben, indaziflam and C17. This indicates thatCellulosin has different target site as isoxaben, indaziflam and C17.Cellulosin has synergistic effect with isoxaben, a benzamide family ofherbicide for preemergence control of broadleaf weeds, and indaziflam.

We have discovered that 250 nM Cellulosin, 4 nM isoxaben or 0.06 nM ofindaziflam did not inhibit plant growth. However, combined application250 nM Cellulosin with 3.5 nM isoxaben and 250 nM Cellulosin with 0.06nM indaziflam significantly inhibits plant growth. These results showthat combined application of Cellulosin with other two herbicides at lowconcentration can be efficient in weed control. This method of herbicideapplication has at least two advantages. First, it reduces the cost ofherbicides because lower dosage of each is needed. Second, becauseCellulosin has different mode of action than isoxaben and indaziflam,combined application of Cellulosin with either of the other two canreduce the chance of herbicide resistance development in weeds that iscaused by repetitive application of the same herbicide. We would like tofile a disclosure on the method of applying Cellulosin together withisoxaben or Cellulosin with indaziflam at the same time for moreefficient weed control.

Cellulosin, Isoxaben, Indaziflam and C17 have Different Mode of Action.

Isoxaben was found as a potent herbicide that targets the cellulosesynthase of plants. Previously, some Arabidopsis cellulose synthasemutants have been found to be resistant to isoxaben (Heim, D. R. et al.,Plant Physiol. 1989, 90, 146-150: Scheible, W. R., et al., Proc NatlAcad Sci USA, 2001, 98, 10079-10084; Desprez, T. et al., Plant Physiol2002, 128, 482-490). These mutants carry mis-sense mutations at theamino acids located at the C-terminus of CesA3 and CesA6. Indaziflam wasrecently characterized as a cellulose biosynthesis inhibitor and hasbeen used as an herbicide (Brabham, C. et al., Plant Physiol 2014, 166,1177-1185). C17 has also been found to inhibit cellulose biosynthesisand has potential to serve as an herbicide (Hu, Z. et al., Plant Physiol2019, 180, 827-836).

Recently, we discovered a new cellulose synthase inhibitor Cellulosinand a group of cesa6 mutants that carry mis-sense mutations at thecentral cytoplasmic region that contains the catalytic motif and areresistant to Cellulosin. In order to test whether Cellulosin has thesame mode of action as isoxaben, indaziflam and C17, we first tested thegrowth of isoxaben resistant mutants on media supplemented withCellulosin. It was discovered that isoxaben resistant mutants ixr1-1,ixr1-2 and ixr2-1 have the same sensitivity to Cellulosin as wildtypeArabidopsis in growth (FIGS. 1A-1F). We next tested the growth of ourCellulosin resistant mutants on isoxaben, indaziflam and C17. It wasfound that while all of these mutants are resistant to Cellulosin, onlya few of our mutants show reduced sensitivity to isoxaben, indaziflamand C17 (FIG. 2 ). Further testing the growth of transgenic plants thatexpress CesA6 with mis-sense mutations at the hypothesized Cellulosinbinding sites in cesa6 loss-of-function mutant background on isoxaben,indaziflam and C17 revealed that these mutants are resistant toCellulosin, they have the same sensitivity to isoxaben, indaziflam andC17 (FIGS. 3A-3D). The results indicate that Cellulosin has a differentmode of action than isoxaben, indaziflam and C17. These results indicatethat Cellulosin has a different mode of action than isoxaben, indaziflamand C17.

Cellulosin has Synergistic Effect with Isoxaben, Indaziflam and C17 inInhibiting Plant Growth.

Because Cellulosin has different target as isoxaben, indaziflam and C17.Since the need to avoid the development of herbicide-resistant weedsthat is caused by repetitive application of the same herbicide isutmost, we decided to test whether application of Cellulosin togetherwith isoxaben, indaziflam and C17 would make it possible to control theweeds more efficiently with reduced dosage of two herbicides. Testingthe growth of Arabidopsis at lower dosage of Cellulosin, isoxaben,indaziflam and C17, we discovered that at 0.25 μM concentration,Cellulosin does not significantly inhibit plant growth (FIGS. 4A-4B). At4 nM concentration, isoxaben does not significantly inhibit plantgrowth. At 0.06 nM, indaziflam does not significantly inhibit plantgrowth. However, when we apply 0.25 μM Cellulosin and 4 nM isoxaben inthe growth media, plant growth is significantly inhibited (FIGS. 4A-4B).When we apply 0.25 μM Cellulsoin and 0.25 nM indaziflam in the growthmedia, plant growth is significantly inhibited as well. This indicatesthat the combination of Cellulosin with isoxaben or indaziflam canincrease the efficiency of weed control.

Cellulosin Inhibits the Growth of Different Plant Species.

In order to test the inhibitory effect of Cellulosin on different plantspecies, we grew different plants in various concentrations ofCellulosin. We found that Cellulosin inhibits tomato and tobacco growthmost efficiently and inhibition was observed with concentrations lessthan 5 μM (FIG. 5A, 5B). Cellulosin significantly inhibited the lawngrasses Kentucky Bluegrass and Perennial Ryegrass at the concentrationof 50 μM (FIG. 5C, 5D). Cellulosin also inhibits the growth of cropssoybean and maize at 50 μM (FIG. 5E, 5F). These results indicate thatCellulosin can inhibit the growth of both dicotyledon and monocotyledonplants and thus can serve as a potential herbicide.

Cellulosin Inhibits Plant Growth when Sprayed on Seedlings.

In order to further test the possibility of using Cellulosin as anherbicide in the field, we sprayed Cellulosin solution on Arabidopsisseedlings grown in the soil. We found that Cellulosin inhibitsArabidopsis growth after being sprayed on the plants (FIGS. 6A-6B). Thisindicates that Cellulosin can be used to control plant growth bydirectly spraying in the soil.

FIGS. 1A-1F show that known isoxaben resistant mutants ixr1-1, ixr1-2and ixr2-1 are not resistant to Cellulosin. Wildtype (WT) Arabidopsisand its mutants ixr1-1, ixr1-2, and ixr2-1 are grown on mediasupplemented with DMSO (FIGS. 1A-1B), isoxaben (FIGS. 1C-1D) orCellulosin (FIGS. 1E-1F) and their quantifications on their root length.Arabidopsis mutants ixr1-1, ixr1-2 and ixr2-1 have longer roots incompare with wildtype (Col) on media supplemented with isoxaben.However, these mutants do not show difference in growth in compare withwildtype (Col) on media supplemented with Cellulosin. Bars: 1 cm

FIGS. 2A-2D show that, as compared with DMSO control (FIG. 2A), most ofCellulosin resistant mutants are sensitive to compounds isoxaben (FIG.2B), indaziflam (FIG. 2C) and C17 (FIG. 2D).

FIGS. 2E-2G depict quantifications of root length of Cellulosinresistant mutants on media supplemented with isoxaben (FIG. 2E),indaziflam (FIG. 2F), and C17 (FIG. 2G) in comparison with DMSO control,Statistically significant differences in right panels were determined byone-way ANOVA test followed by Tukey's multiple comparisons test.Different letters of root length of seedlings indicate significantdifference between groups (p<0.05). Bars: 1 cm.

FIGS. 3A-3D demonstrate that plants expressing CesA6 carrying mis-sensemutations at the catalytic site of Cellulosin do not show resistance toisoxaben, indaziflam and C17 in growth compared with DMSO control: DMSO(FIG. 3A), isoxaben (FIG. 3B), Indaziflam (FIG. 3C) and C17 (FIG. 3D).Bars: lcm.

FIGS. 4A-4B demonstrate the synergistic effect of Cellulosin withisoxaben and indaziflam. FIG. 4A shows Arabidopsis wildtype plants grownon DMSO, Cellulsoin, isoxaben, indaziflam, C17, Cellulosin withisoxaben, Cellulosin with indaziflam, and Cellulosin with C17. Plantgrowth was not inhibited by 0.25 μM Cellulosin, 4 nM isoxaben, 0.25 nMindaziflam or 4 nM C17 significantly. However, combined application of0.25 μM Cellulosin and 4 nM isoxaben, 0.25 μM Cellulosin and 0.25 nMindaziflam, or 0.25 μM Cellulosin and 4 nM C17 significantly inhibitedplant growth. Bar, lcm.

FIG. 4B shows quantification on the root length of Arabidopsis plantsgrown on media with control (0.1% DMSO), 0.25 μM Cellulosin, 4 nMisoxaben, 0.25 nM indaziflam, 4 nM C17, 0.25 μM Cellulosin and 4 nMisoxaben, 0.25 μM Cellulosin with 0.25 nM indaziflam, or 0.25 μMCellulosin and 4 nM C17. Statistically significant differences in rightpanels were determined by one-way ANOVA test followed by Tukey'smultiple comparisons test. Different letters of root length of seedlingsindicate significant difference between groups (p<0.05). N=12.

FIGS. 5A-5F demonstrate that Cellulosin inhibits the growth of differentplant species. FIG. 5A shows Cellulosin inhibition of tomato growth.FIG. 5B shows Cellulosin inhibition of tobacco growth. FIG. 5C showsCellulosin inhibition of Kentucky Bluegrass growth. FIG. 5D showsCellulosin inhibition of Perennial Ryegrass growth. FIG. 5E showsCellulosin inhibition of soybean growth. FIG. 5F shows Cellulosininhibition of maize growth. Bars: 1 cm. Statistical analysis wasperformed using student t-test. ** p<0.01. *** p<0.001.

FIGS. 6A-6B demonstrate that Cellulosin inhibits plant growth in soil.FIG. 6A shows Arabidopsis plants sprayed with control solution. FIG. 6Bshows Arabidopsis plants sprayed with Cellulosin (500 μM).

FIG. 7 demonstrates synergistic effect between Cellulosin and isoxaben.Neither 300 nM Cellulosin nor 3.5 nM isoxaben significantly inhibitsplant growth. However, combined application of 300 nM and 3.5 nMisoxaben significantly inhibits plant growth. Nine days old Arabidopsisplants grown on control condition (0.1% DMSO), 300 nM Cellulosin, 3.5 nMisoxaben, or 300 nM Cellulosin and 3.5 nM Isoxaben are shown. Bar, lcm.

Cellulosin and isoxaben have synergistic effect in inhibiting plantgrowth. Because Cellulosin and isoxaben target CesA at different sites,it allowed us to hypothesize that application of both herbicides at thesame time will make it possible to control the weeds with reduced dosageof both herbicides. This should also avoid the development ofherbicide-resistant weeds that is caused by repetitive application ofthe same herbicide. We tested the growth of Arabidopsis at lower dosageof both Cellulosin and isoxaben. It was found that Cellulosin does notsignificantly inhibit plant growth at 300 nM concentration; and at 3.5nM concentration, isoxaben does not significantly inhibit plant growtheither. However, when we apply 300 nM Cellulosin and 3.5 nM isoxaben inthe growth media, plant growth is significantly inhibited (FIG. 7 ).This indicates that the combination of both Cellulosin and isoxabenherbicides can increase the efficiency of weed control.

In summary, Cellulosin has a different mode of action than isoxaben,indaziflam and C17. Cellulosin inhibits the growth of different plantspecies, which makes it a broad-spectrum herbicide. Cellulosin hassynergistic effects with isoxaben, indaziflam and C17. Application ofCellulosin together with isoxaben, indaziflam or C17 increases theefficiency and long term effects of weeds control. Cellulosin inhibitsplant growth condition in soil, which indicates that it can be used as aherbicide to control weeds growth by being sprayed.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. It should be understood by those skilled inthe art that various alternatives to the embodiments described hereinmay be employed in practicing the claims without departing from thespirit and scope as defined in the following claims.

What is claimed is:
 1. A composition comprising isoxaben and cellulosin,together with one or more diluents, excipients or carriers.
 2. Thecomposition of claim 1, further comprising one or more additionalherbicides.
 3. The composition of claim 1, wherein the ratio of isoxabenand cellulosin ranges from about 1:100 to about 100:1 on a molar basis.